Heparin-independent, high sensitivity platelet function evaluation technique

Abstract

An method for performing activated clotting time tests, including a method for evaluating platelet functionality of a blood sample. The method includes the steps of combining a heparin-inactivating agent, an anticoagulant agent, a sufficient amount of clotting reagent to achieve clotting, a platelet activating agent, and the sample of blood to be tested to form a test mixture. The platelet activating agent is a reagent other than the clotting reagent. The platelets of the sample are activated by agitating the test mixture, and the activated clotting time test is terminated upon detecting a predetermined change in a property of the test mixture. The activated clotting time of the sample of blood is calculated based on the elapsed time. The activated clotting time test may be performed using a plunger sensor apparatus which comprises a plurality of test cells. In this method, each of the cells of the apparatus includes a heparin-inactivating agent, an anticoagulant agent, and a sufficient amount of a clotting reagent to achieve clotting. At least one of the test cells further includes a platelet activating agent, which is a reagent other than the clotting reagent. The clotting time is determined for each of the aliquot portions, and the relative clotting times of the aliquot portions in the cells are determinative of the platelet functionality of the sample.

We claim: 
     
       1. A method for performing an activated clotting time test on a sample of blood containing platelets, said method comprising: combining a heparin-inactivating agent, and anticoagulant agent, a sufficient amount of a clotting reagent to achieve clotting, a platelet activating agent, and the sample of blood to be tested to form a test mixture at the start of the activated clotting time test, wherein said platelet activating agent is a reagent other than said clotting reagent;   activating the platelets of the sample by agitating the test mixture;   terminating the activated clotting time test upon detecting a predetermined change in a property of the test mixture;   measuring an elapsed time from the start of the activated clotting time test to the termination of the clotting time test; and   calculating the activated clotting time of the sample of blood based on the elapsed time.   
     
     
       2. The method as defined in claim 1, wherein the heparin-inactivating agent is heparinase. 
     
     
       3. The method as defined in claim 1, wherein the heparin-inactivating agent is present at a concentration of between about 0.1 and about 10 international units per milliliter of blood sample. 
     
     
       4. The method as defined in claim 1, wherein the heparin-inactivating agent is present at a concentration of between about 1.0 and about 6.0 international units per milliliter of blood sample. 
     
     
       5. The method as defined in claim 1, wherein the heparin-inactivating agent is present at a concentration of between about 1.5 and about 2.5 international units per milliliter of blood sample. 
     
     
       6. The method as defined in claim 1, wherein the anticoagulant agent is an inhibitor of at least one of Factor Xa and Factor II. 
     
     
       7. The method as defined in claim 1, wherein the anticoagulant agent is a substrate-derived competitive thrombin inhibitor. 
     
     
       8. The method as defined in claim 1, wherein the anticoagulant agent is a thrombin inhibitor selected from the group consisting of synthetic peptides, arginine derivatives, benzamidine derivatives, and lysine derivatives. 
     
     
       9. The method as defined in claim 1, wherein the anticoagulant agent is argatroban. 
     
     
       10. The method as defined in claim 1, wherein the anticoagulant agent is present at a concentration of between about 0.1 μg and about 20 μg per milliliter of blood sample. 
     
     
       11. The method as defined in claim 1, wherein the anticoagulant agent is present at a concentration of between about 1 μg and about 15 μg per milliliter of blood sample. 
     
     
       12. The method as defined in claim 1, wherein the anticoagulant agent is present at a concentration of between about 7 μg and about 12 μg per milliliter of blood sample. 
     
     
       13. The method as defined in claim 1, wherein the clotting reagent is an activator of at least one of Factor XII and Factor XI. 
     
     
       14. The method as defined in claim 1, wherein the clotting reagent is kaolin or diatomaceous earth. 
     
     
       15. The method as defined in claim 1, wherein the clotting reagent is kaolin. 
     
     
       16. The method as defined in claim 1, wherein the platelet activating agent is selected from the group consisting of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, arachidonic acid, collagen, epinephrine, and ristocetin. 
     
     
       17. The method as defined in claim 1, wherein the platelet activating agent is 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine. 
     
     
       18. The method as defined in claim 1, wherein the platelet activating agent in the blood sample is present at a concentration of between 0 and about 10 μM. 
     
     
       19. The method as defined in claim 1, wherein the platelet activating agent in the blood sample is present at a concentration of between 0 and about 1.0 μM. 
     
     
       20. The method as defined in claim 1, wherein the platelet activating agent in the blood sample is present at a concentration of between 0 and about 200 nM. 
     
     
       21. The method as defined in claim 1, wherein the activated clotting time test is performed using a plunger sensor technique. 
     
     
       22. The method as defined in claim 1, wherein the blood sample comprises a therapeutic amount of a platelet function inhibitor. 
     
     
       23. The method as defined in claim 1, wherein said predetermined change in a property of the test mixture is a change in viscosity of said test mixture. 
     
     
       24. The method as defined in claim 22, wherein the platelet function inhibitor is selected from the group consisting of Abciximab, 4-[4-[4-(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidineacetic acid hydrochloride trihydrate and acetylsalicylic acid. 
     
     
       25. The method as defined in claim 22, wherein the platelet function inhibitor is Abciximab and wherein the platelet activating agent is 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine. 
     
     
       26. The method as defined in claim 22, wherein the platelet function inhibitor is 4-[4-[4-(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidineacetic acid hydrochloride trihydrate and wherein the platelet activating agent is 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine. 
     
     
       27. The method as defined in claim 22, wherein the platelet function inhibitor is acetylsalicylic acid and wherein the platelet activating agent is arachidonic acid. 
     
     
       28. A method for performing an activated clotting time test on a sample of blood containing platelets using a plunger sensor apparatus, said apparatus comprising at least one test cell and a plunger assembly within said test cell, said method comprising: combining in said at least one test cell of said plunger sensor apparatus a heparin-inactivating agent, and anticoagulant agent, a sufficient amount of a clotting reagent to achieve clotting, and a platelet activating agent, wherein said platelet activating agent is a reagent other than said clotting reagent;   dispensing said sample of blood into the test cell to form a test mixture;   reciprocating the plunger assembly in the test mixture by alternately lifting the plunger assembly and allowing the plunger assembly to descend through the test mixture;   detecting a point in time at which a predetermined property of the test mixture changes by a predetermined expected amount by sensing the descent of the plunger assembly, the predetermined property affecting the activated clotting time test;   measuring an elapsed time from the beginning of the step of reciprocating the plunger assembly in the test mixture to the point in time at which the predetermined property of the test mixture changes by the predetermined expected amount; and   calculating the activated clotting time of the sample of blood based on the elapsed time.   
     
     
       29. The method as defined in claim 28, wherein at least one of said test cells has no platelet activating agent. 
     
     
       30. The method as defined in claim 28, wherein the heparin-inactivating agent is heparinase. 
     
     
       31. The method as defined in claim 28, wherein the heparin-inactivating agent is present at a concentration of between about 1.0 and about 6.0 international units per milliliter of blood sample. 
     
     
       32. The method as defined in claim 28, wherein the anticoagulant agent is a thrombin inhibitor selected from the group consisting of synthetic peptides, arginine derivatives, benzamidine derivatives, and lysine derivatives. 
     
     
       33. The method as defined in claim 28, wherein the anticoagulant agent is argatroban. 
     
     
       34. The method as defined in claim 28, wherein the anticoagulant is present at a concentration of between about 0.1 μg and about 20 μg per milliliter of blood sample. 
     
     
       35. The method as defined in claim 28, wherein the clotting reagent is an activator of at least one of Factor XII and Factor XI. 
     
     
       36. The method as defined in claim 28, wherein the clotting reagent is kaolin or diatomaceous earth. 
     
     
       37. The method as defined in claim 28, wherein the platelet activating agent is selected from the group consisting of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, arachidonic acid, collagen, epinephrine and ristocetin. 
     
     
       38. The method as defined in claim 28, wherein the platelet activating agent is 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine. 
     
     
       39. The method as defined in claim 28, wherein the platelet activating agent is present at a concentration of between 0 and about 10 μM. 
     
     
       40. The method as defined in claim 28 wherein said predetermined property is a change in viscosity of said test mixture.